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Issued  January  11,  1909. 


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U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BuLi.F.TiN  111. 
A.  I).  MEL,VIJHh  Chief  of  Bureau. 


CHEMICAL  AND  PHYSICAL  STUDY  OF 
THE  LARGE  AND  SMALL  FAT 
GLOBULES  IN  COWS'  MILK. 


BY 


R.  H.  SHAW, 
Assistant  /dairyman.  Dairy  Division, 

AND 

C.   H.  ECKLES. 
Prdffssor  of  Dairy  Husbandry,  University  of  Missouri. 


Ur<;v::.:-..iY  OF  CALJFOar-iiA- 
LOS  ANGELES 

SEP  23  1952 

LIBRARY 
GOVT.  PUBS.  ROOM 


WASHINGTON: 

GOVERNMENT    PRINTING    OFFICE. 

190^). 


Digitized  by  tine  Internet  Arciiive 
in  2007  with  funding  from   . 
IVIicrosoft  Corporatipn. 


http://www.arcliive.org/details/chemibalplTygicaJOOsliawiala 


Issued  January  11,  1909. 

U.  S.  DEPARTMENT  OF  AGRICULTURE. 

BUREAU  OF  ANIMAL  INDUSTRY— Bulletin  1t1. 

A.  D.  MELVIN,  Chief  of  BuRtAU. 


A  CHEMICAL  AND  PHYSICAL  STUDY  OF 

THE  LARGE  AND  SMALL  FAT 

GLOBULES  IN  COWS'  MILK. 


BY 


R.  H.  SHAW. 
Assistant  Dairyman,  Dairy  Division, 

AND 

C.   H.  ECKLES, 
Professor  of  Dairy  Husbandry,  University  of  Missouri. 


WASHINGTON: 
GOVERNMENT    PRINTING    OFFICE. 

1909. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 

Chic/:  A.  D.  MF.I.VIN. 

Assistant  Chief:  A.  M.  Farrixgton. 

Chief  Clerk:  Charles  C.  Carroll. 

Biochemic  Difision:  M.  Dorset,  chief;  James  A.  Emery,  a^sistaut  chief. 

Dainf  Division:  Ed.  H.  Webster,  chief;  C.  B.  Lane,  assistant  chief . 

Inspection  Division:  Rice  P.  Steddom,  chief;  Morris  Wooden,  R.A.Ramsay, 
and  Albert  E.  Behnke,  associate  chiefs. 

Pathological  Division:  John  R.  Mohler,  chief;  Henry  J.  Washburn,  assistant 
chief. 

Quarantine  Dii'ision:  Richard  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schroeder,  superintendent;  W.  E.  Cotton,  as-sistant. 

Animal  Husbandman:  George  M.  Rommel. 

Editor:  James  M.  Pickens. 

DAIRY  DIVISION. 
Chief:  Ed.  H.  Webster. 

Assistant  Chi^f:  C.  B.  Lane. 

Librarian:  Miss  C.  B.  Sherman. 

dairy  farming  investigations. 

Assistant  in  charge,  B.  JJ.  Rawl;  assistant,  Duncan  Stuart. 

Dairy  buildings:  J.  A.  Conover;  architect,  K.  E.  Parks;  ventilation  experiment", 
C.  R.  Potteiger. 

Herdbook  work:  Helmer  Rabild  and  William  Hart  Dexter. 

Southern  dairying:  S.  E.  Barnes,  J.  E.  Dorman,  J.  T.  Eaton,  H.  P.  Lykes,  J.  H. 
McClain,  A.  K.'jlisser,  H.  R.  Welch,  and  T.  R.  Woodward. 

dairy  products  investigations.  , 

Assistant  in  charge,  L.  A.  Rogers. 

Butter  investigations,  Albert  Lea,  Minn.,  and  Washington,  D.  C:  Chemist,  W.  N. 
Berg;  bacteriorologist,  S.  H.  Ayers. 

Swiss  cheese  investigations,  Albert  Lea,  Minn.:  In  charge,  C.  F.  Doane;  assistant, 
T.  W.  Issajeff. 

Cheese  investigations,  Madison,  Wis. :  Chemist,  S.  K.  Suzuki;  bacteriologist,  Alfred 
Larson;  cheese  maker,  J.  W.  Moore. 

Cheese  investigations,  Storrs,  Conn.:  Mycologist,  Charles  Thom;  chemist,  Arthur 
W.  Dox. 

Milk  secretion  investigations,  Columbia,  Mo.:  Chemist,  R.  H.  Shaw;  assistants, 
J.  O.  Halverson,  A.  E.  Perkins,  and  G.  C.  Payne. 

DAIRY   manufacturing    INVESTIGATIONS. 

Assistant  in  charge,  B.  D.  White;  assistant,  S.  C.  Thompson. 

Creamery  records,  Albert  Lea,  Minn.:  Creamery  practice,  John  L.  Sherk;  assistants 
(collaborators),  P.  W.  Noble  and  J.  D.  Burk. 

Creamery  practice  investigations:  J.  C.  Joslin,  Robert  McAdam,  F.  L.  Odell,  J.  C. 
Winkjer,  and  Thomas  Corneliuson. 

Market  investigations:  New  York  City,  C.  W.  Fryhofer;  Chicago,  H.  J.  Credicott; 
San  Francisco,  C.  L.  Mitchell. 

market   milk   INVESTIGATIONS. 

Assistant  chief  of  division  in  charge,  a^isistants,  Lee  H.  P.  Maynard,  Ivan  C.  Weld, 
and  George  M.  Whitaker. 

RENOVATED    BUTTER    INSPECTION-. 

Thief  in=ipector,  M.  W.  Lang,  Chicago;  assistant,  Lt'vi  Wells,  Now  York. 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 
Washington,  D.  C,  October  W,  190S. 
Sir:  I  have  the  honor  to  transmit  herewith,  and  to  recommend  for 
pubhcation  in  the  bulletin  series  of  this  Bureau,  the  accompanyinti: 
manuscript  of  an  article  entitled  "A  Chemical  and  Physical  Study  of 
the  Large  and  Small  Fat  Globules  in  Cows'  Milk,"  by  R.  H.  Shaw,  of  the 
Dairy  Division  of  this  Bureau,  and  C.  11.  Eckles,  of  the  Missouri 
Agricultural  Experiment  Station.  This  work  is  a  preliminary  part 
of  a  comprehensive  investigation  being  conducted  at  the  Missouri 
station  by  cooperation  between  that  station  and  the  Dairy  Division 
concerning  the  eflfects  of  period  of  lactation,  feed,  and  other  factors 
on  the  chemical  composition  of  milk. 

At  the  request  of  the  authors  acknowledgment  is  made  to  Messrs. 
J.  O.  Ilalverson  and  G.  C.  Payne  for  assistance  in  the  chemical  and 
microscopical  work. 

Respectfully,  A.  D.  Melvin, 

Cliief  of  Bureau . 
Hon.  James  Wilson, 

Secretary  of  Agriculture. 


CONTENTS. 


Pace. 

Introtluction 5 

Previous  inveetigations 5 

Experimental  work 8 

Source  of  the  samples 8 

Method  of  separation 8 

Preparation  of  fat  samples  for  analysis 10 

Analytical  methods 10 

Color  determinations 11 

Microscopical  work 11 

Results  of  experiments 14 

Conclusion 16 


ILLUSTRATIONS. 


Page. 

Fig.  1 .  Showing  method  of  counting  globules 12 

2.  Showing  the  average  relative  size  of  large  and  small  fat  globules 13 


A  CHEMICAL  AND  PHYSICAL  STUDY  OF  THE  LARGE 
AND  SMALL  FAT  GLOBULES  IN  COWS'  MILK. 


INTRODUCTION. 

•  In  planning  a  long  investigation  on  the  effect  of  the  period  of 
lactation,  feed,  and  other  factors  on  the  chemical  composition  of 
milk  it  was  decided  that  the  most  practical  way  of  extracting  the  fat 
from  the  milk  was  by  passing  it  through  a  small  separator  and 
churning  the  cream.  In  this  method  the  tendency  is  for  the  smaller 
globules  to  escape  in  the  skim  milk  and  buttermilk ;  hence  it  became 
necessary  to  ascertain  whether  or  not  these  small  globules  which  in 
part  escape  differ  in  chemical  composition  from  the  large  globules, 
and  if  such  a  difference  exists,  to  determine  just  what  it  is. 

Investigations  on  these  points  are  not  lacking,  and  had  these  inves- 
tigations been  sufficiently  extensive,  or  had  they  agreed  on  the  prin- 
cipal points,  the  work  herein  presented  would  have  been  unnecessary. 
However,  such  is  not  the  case.  There  is  almost  a  total  lack  of  agree- 
ment in  the  work  hitherto  published,  as  will  be  seen  later  on.  When 
the  present  investigation  was  taken  up  it  was  the  intention  to  cover 
only  those  points  necessary  to  valitlate  certain  determinations 
involved  in  the  long  investigation  mentioned  above.  Later  it  was 
decided  to  enlarge  it  to  some  extent  and  publish  it  as  a  contribution 
to  the  knowledge  of  the  chemistry  of  butterfat. 

PREVIOUS  INVESTIGATIONS. 

Miiller"  (1867)  observed  that  fat  extracted  from  skimmed  milk  is 
waxlike,  and  also  that  in  the  analysis  of  butter  where  100  grams  or 
more  of  fat-free  substance  is  obtained  the  last  particles  of  fat 
extracted  have  more  the  properties  of  wax  than  of  butterfat. 

Schroeder''  in  1872  published  some  investigations  showing  that 
when  milk  is  fractionally  creamed  the  butterfat  obtained  from  the 

"MQller,  Alexander.  Chemische  untersuchungen  auf  dem  gebiete  der  milch- 
wirtlischaft.  Die  Landwirthflchaftlichen  Versuchs-Stationen,  Band  9,  pp.  364-396. 
See  p.  390.     Chemnitz,  1867. 

^Schrueder,  G.  Oeitrage  zur  kenntnissdes  fettgehalts  der  milch.  Milch  Zeitung, 
No.  28.  pp.  32&-327,  1872. 

6 


6  LARGE   AND   SMALL   FAT   GLOBULES    IN    cOWS'  MILK. 

first  fraction  has  a  lower  ineltinj;  point  ami  lower  specific  gravity  than 
the  butterfat  obtained  from  the  last  fraction.  He  also  stated  that  the 
latter  contained  no  butyric  acid. 

Klusemann"  in  1893  conducted  an  investigation  with  milk  from 
individual  cows.  He  separated  the  large  and  small  fat  globules  by 
passing  the  whole  milk,  at  a  temperature  of  5  to  12°  C,  through  a  De 
Laval  separator  running  at  low  speed.  The  cream  contained  the  large 
globules.  The  skim  milk  was  then  warmed  somewhat  and  again 
passed  through  the  separator  running  more  rapidly.  The  cream  in 
this  instance  contained  the  medium-sized  globules.  The  small  glob- 
ules were  obtained  by  heating  the  last  skim  milk  to  36  to  38°  C.  and 
again  passing  it  through  the  separator,  this  time  running  very  rap- 
idly; the  cream  was  churned  in  a  small  dash  chum,  and  the  resulting 
butter  was  melted  and  filtered  by  means  of  a  jacketed  filter. 

He  determined  the  melting  and  freezing  points,  the  water-insoluble 
fatty  acids,  the  melting  and  freezing  points  of  the  insoluble 
fatty  acids,  the  iodin  absorption  number,  the  volatile  acids,  and  the 
specific  gravity.  In  his  conclusions  he  states  that  the  percentage 
of  the  insoluble  fatty  acids,  and  the  melting  point  and  freezing  point 
of  the  butterfat,  and  of  the  insoluble  fatty  acids  are  higher  in  fat 
from  small-globule  cream  than  in  fat  from  large-globule  cream,  the 
reverse  being  true  of  the  percentage  of  volatile  acids  and  of  the 
iodin  number. 

Gutzeit''  (1895)  published  an  investigation  on  the  same  subject. 
He  chose  mixed  milk  from  a  herd  of  150  cows.  He  separated  the 
large  and  small  globules  as  follows:  He  used  100  liters  of  milk, 
20  liters  of  which  was  allowed  to  rise  for  six  hours  in  a  pan  immersed 
in  cold  water.  The  resulting  cream  contained  the  large  fat  globules. 
The  remaining  80  liters  was  then  run  through  a  separator,  the  cream 
discarded,  and  the  skim  milk  coagulated.  The  whey  was  then  run 
through  a  separator,  and  the  resulting  whey  cream  contained  the 
small  globules.  The  tw^o  creams  were  churned  separately,  and  the 
resulting  butters  w^ere  heated  for  a  time  at  60°  C.  and  filtered.  He 
determined  the  specific  gravity,  the  melting  point,  the  refractive 
index,  insoluble  fatty  acids,  volatile  fatty  acids,  saponification 
number,  and  iodin  absorption  number,  and  concludes  that  in  homo- 
geneous milk  the  fat  globules  of  all  sizes  have  the  same  chemical- 
physical  composition. 

aKlusemaan,  Erich.  Die  zusammensetzung  und  die  beschaffenheit  der  aus  den 
grofisen  und  den  kleinen  fettkflgelchen  der  kuhmilch  gewonnenen  butter.  Inaugural 
dissertation.     Leipzig,  1893. 

I>  Gutzeit,  Ernst.  Die  schwankungen  der  mittleren  groese  der  fettkiigelchen  in 
der  kuhmilch  nach  laktation,  fiitlerung  und  raese,  sowie  fiber  den  physikalischen 
und  chemisohen  unterschied  der  groesten  und  kleinsten  fettkugelchen.  Land- 
•wirthflchaftliche  JahrbQcher,  Bans  24,  pp.  539-668.     Berlin,  1895. 


PREVIOUS   INVESTIGATIO>'S.  7 

Lemus"  (1902)  separated  the  large  and  small  globules  by  inter- 
rupted milking.  The  fu-st  liter  of  milk  drawn  he  took  to  represent 
the  small  globules  and  the  last  fraction  of  about  one-half  liter  drawn 
the  large  globules.  After  reserving  a  sample  for  fat  determination 
and  microscopical  examination,  he  extracted  the  fat  according  to  the 
method  of  Soxhlet  by  shaking  with  ether  after  having  first  made  it 
alkaline  with  potassium  hydroxid.  The  ethereal  layer  was  then 
removed  and  dried  at  100°  C.  to  a  constant  weight.  He  made 
some  of  the  same  determinations  as  did  Klusemann  and  Gutzeit, 
but  in  his  conclusions  refers  only  to  the  volatile  acids  and  the  oleic 
acid.  He  states  that  in  the  majority  of  cases  the  smaller  fat  globules 
contained  more  oleic  acid  and  less  volatile  acids  than  did  the  larger 
globules. 

In  the  three  more  recent  investigations,  that  is,  those  by  Kluse- 
mann, Gutzeit,  and  Ijcmus,  it  is  seen  that  no  two  draw  the  same 
conclusions.  Gutzeit  claims  that  there  is  no  difference,  chemically 
or  physically,  between  the  large  and  small  fat  globules  in  the  same 
milk.  Klusemann  and  I^emus  agree  in  claiming  that  there  is  a  differ- 
ence, but  disagree  on  some  of  the  points  of  difference. 

Klusemann  worked  on  the  milk  of  five  individual  cows  and  on  the 
mixed  milk  of  seven.  A  study  of  his  results  shows  that  in  every  case 
the  percentage  of  insoluble  acids  in  small-globule  fat  is  higher  than  in 
the  large-globule  fat,  the  difference  being  from  about  0.3  per  cent 
to  about  3  per  cent;  the  melting  point  of  the  butterfat  was  higher 
in  the  small-globule  fat  than  in  the  large,  the  variation  being  from 
0.5  to  2°  C. ;  the  volatile  fatty  acids  in  terms  of  Reichert-Meissl 
number  showed  a  decrease  in  the  small-globule  fat  in  six  cases 
and  an  increase  in  two  cases,  the  variation  l)eing  from  about  1  c.  c. 
to  about  4. .5  c.  c;  the  iodin  number  in  three  cases  showed  practically 
no  difference,  in  three  cases  it  showed  a  decrease  in  the  small-globule 
fat  and  in  one  an  increase,  the  largest  difference  being  about  2.5 
per  cent.  Granting  that  no  criticism  can  be  made  of  his  methods 
and  work,  it  would  seem  that  with  the  limited  number  of  animals 
used  his  conclusions  even  on  the  insoluble  fatty  aciils  and  melting 
point  are  unwarranted,  and  certainly  not  justified  in  the  case  of  the 
other  constants. 

In  Lemus's  investigation  it  may  be  seen  that  the  .separation  of  the 
large  and  small  globules  is  not  positive.  In  fact,  instead  of  the 
relative  size  of  the  fat  globules  being  invariably  larger  in  the  stripper 
milk  than  in  the  foremilk,  the  reverse  is  true  in  some  cases.  In  10 
cases  the  iodin  number  was  smaller  in  the  large-globule  fat  than  in 
the  small-globule  fat,  and  in  3  cases  it  was  larger,  the  difference  ranging 

"  Lemufl,  \S'oldemar.  I'fber  die  chc»niL'«-he  b««f»<haff<'nhf>it  dos  in  don  gro.sfnMi  iind 
in  den  kleineDinilehkilgelchencnthaltenenfetteH.  Inauguraldi;<i<«rtation.  Leipzig, 
1902. 


8  LARGE  AND  SMALL  FAT  GLOBULES   IN    COWS'  MILK. 

from  0.11  to  3.54  j)er  cent.  With  the  volatile  acicls  the  Reichert- 
Meissl  number  was  vsmaller  in  16  cases  in  the  large-globule  fat  than 
in  the  small-globule  fat,  and  in  6  cases  the  reverse  obtained,  the  differ- 
ences ranging  from  0.45  to  13.85  per  cent.  It  is  also  doubtful  whether 
the  fat  remained  unchanged  in  the  preparation  of  the  samples,  where 
a  prolonged  heating  at  100°  C.  was  necessary  to  eliminate  the  ether." 
Moreover,  it  would  seem  that  his  determinations  show  the  difference 
betw^een  the  fat  in  the  foremilk  and  that  in  the  milk  secreted  during 
the  process  of  milking,  rather  than  that  between  the  large  and  small 
fat  globules  in  the  same  milk. 

EXPERIMENTAL  WORK. 

For  our  investigation  animals  were  selected  which  exhibited  a  wide 
range  of  breed,  age,  and  period  of  lactation.  In  the  earlier  part  of  the 
investigation  the  chemical  and  microscopical  work  was  limited  to 
determinations  of  the  relative  size  of  the  fat  globules,  the  iodin  num- 
ber, the  Reichert-Meissl  number,  the  Koetstorfer  number,  and  the 
refractive  index.  Later  it  was  extended  to  include  the  specific 
gravity,  the  melting  point,  the  Hehner  number,  the  color,  and  the 
percentage  of  different-sized  fat  globules. 

Table  1  gives  the  data  concerning  the  animals.  In  Tables  2,  3, 
and  4  vriW  be  found  the  results  of  the  chemical  and  microscopical 
work. 

SOURCE    OF   THE    SAMPLES. 

The  cows  selected  to  supply  the  samples  represented  four  breeds — 
Jersey,  Holstein-Friesian,  Ayrshire,  and  Shorthorn — and  were  mem- 
bers of  the  Missouri  Agricultural  College  herd.  Groups  supplying 
the  samples  were  arranged  with  a  view  of  having  certain  samples 
represent  milk  from  cows  in  the  early  part  of  the  period  of  lactation, 
while  others  represented  milk  produced  in  the  latter  part  of  the  period. 
The  table  shows  the  number  of  days  in  milk,  the  breed,  the  age,  the 
date  at  which  samples  were  taken,  and  the  average  percentage  of  fat 
in  the  milk  of  each  cow.  The  sample  of  milk  taken  was  the  total 
product  for  one  day  of  the  group  of  cows  shown  in  the  table. 

METHOD   OF   SEPARATION. 

The  first  method  tried  consisted  in  allowing  the  milk  to  stand  at 
room  temperature  for  about  two  hours  until  a  portion  of  the  cream 
had  come  to  th^  surface,  when  it  was  removed  by  drawing  the  remain- 
der of  the  milk  from  a  faucet  in  the  bottom  of  the  can.     The  partially 

0  According  to  C.  A.  Brown  (Annual  Report  of  the  Penney Ivania  State  College, 
1899-1900.  p.  208),  a  temperature  much  above  50°  C.  will  very  eoon  alter  the  compo- 
sition of  butterfat.  Butterfat  kept  at  50°  C.  for  two  days  showed  a  loss  of  over  one 
unit  in  iodin  number. 


EXPERIMENTAL   WORK.  9 

skimmed  milk  secured  in  this  way  was  again  placed  in  the  can  and 
allowed  to  stand  about  twelve  hours,  when  the  second  portion  of  the 
cream  was  removed  as  before.  This  skim  milk,  which  still  contained 
approximately  0.3  per  cent  of  fat,  was  then  run  through  a  centrifugal 
separator  at  a  temperature  of  32°  C.  The  cream  thus  secured  contained 
the  smallest  fat  globules  which  it  is  possible  to  obtain  by  the  centrifugal 
machine.  The  cream  secured  from  the  first  skimming  contained  the 
larger  fat  globules,  as  they  reach  the  surface  first  under  the  influence 
of  gravity,  but  it  also  contained  some  small  fat  globules.  In  order 
to  eliminate  these  this  cream  was  placed  in  the  bottom  of  a  deep, 
narrow  can,  which  was  then  filled  with  the  skim  milk  from  the  final 
separation  with  the  centrifugal  separator.  When  most  of  the  cream 
had  again  risen  it  was  skimmed  off  and  retained  for  the  sample  of  the 
large  fat  globules. 

The  above  method  was  found  to  be  satisfactory,  but  equally  good 
results  were  secured,  at  a  considerable  saving  of  time,  by  using  the 
centrifugal  separator.  After  several  preliminary  trials  the  following 
plan  was  adopted: 

The  milk  was  first  run  through  the  centrifugal  separator  at  a  tem- 
perature of  30°  to  32°  C.  and  the  speed  of  the  machine  so  regulated 
by  trial  that  approximately  one-half  of  the  fat  in  the  original  milk 
was  taken  out  as  cream  and  one-half  remained  in  the  skim  milk.  It 
was  found  that  with  a  hand-power  separator,  the  normal  speed  of 
which  was  60  revolutions  of  the  crank  per  minute,  this  result  was 
obtained  when  the  machine  was  operated  at  the  rate  of  15  to  25  revo- 
lutions a  minute,  depending  upon  the  character  of  the  milk  separated. 
The  cream  secured  from  this  separation  contained  the  larger  fat  glob- 
ules, but  also  a  large  number  of  medium  sized  and  some  small  ones, 
which  were  removed  as  described  later. 

The  skim  milk  secured  by  this  separation  contained  approximately 
one-half  the  fat  originally  found  in  the  milk,  and  was  again  separated 
at  a  higher  speed — from  29  to  40  revolutions  of  the  crank  to  the 
minute.  The  speed  was  adjusted  after  trial  with  each  sample  of 
milk  so  that  about  0.2  ]>er  cent  of  fat  remaine<l  in  the  skim  milk. 
The  cream  from  this  second  separation  was  rejected,  as  it  contained 
medium  sized  and  some  large  fat  globules  which  had  escaped  the  first 
separation. 

The  skim  milk  containing  approximately  0.2  per  cent  of  fat  was 
then  heated  to  38°  C.  and  again  nm  through  the  centrifugal  .separator 
at  a  speed  of  from  65  to  70  revolutions  of  the  crank  to  the  minute. 
The  cream  from  this  sej)aration  contained  the  smallest  fat  globules 
which  it  is  possible  to  secure  with  a  centrifugal  separator.  It  is 
impossible  to  obtain  all  of  the  fat  by  any  mechanical  method  of 
separation.  The  fat  remaining  in  the  skim  milk  after  this  final 
separation  was  found  to  be  from  0.025  to  0.004  per  cent,  as  shown  by 
the  Babcock  method,  using  the  double-necked  Wagner  testing  bottle. 


10  LARGE   AND   SMALL   FAT   GLOBULES   IN    COWS'  MILK. 

The  cream  from  the  first  separation  contaiiiint]:  the  largest  fat 
.  globules  was  added  to  the  skim  milk  from  the  third  and  run  through 
the  separator  again  at  the  s|)eed  used  in  the  first  separation,  to 
eliminate  the  smaller  fat  globules  which  had  remained  with  the  large. 
The  cream  from  tliis  separation  was  again  niLxed  with  a  second  lot 
of  the  same  skim  milk  from  the  third  separation  and  the  process 
repeated,  this  cream  being  taken  as  the  final  sample  containing  the 
largest  fat  globules.  The  two  lots  of  cream  were  then  churned  by 
shaking  in  a  closed  jar  as  long  as  any  butter  could  be  accumulated. 

PREPARATION  OF  FAT  SAMPLES  FOR  ANALYSIS. 

The  butter  was  immediately  melted  on  a  steam  bath  and  allowed 
to  remain  in  this  condition  until  the  curd  and  water  had  settled. 
Special  care  was  taken  at  this  point  that  the  temperature  did  not  rise 
above  50°  or  55"  C.  and  also  that  it  remained,  even  at  this  tempera- 
ture, no  longer  than  was  necessary.  It  was  then  filtered  through  a 
paper  filter  kept  warm  by  an  electrical  device,  and  preserved  in 
corked  bottles  which  were  protected  from  light  in  a  refrigerator  until 
the  butter  was  analyzed. 

ANALYTICAL   METHODS. 

The  methods  of  the  Association  of  Official  Agricultural  Chemists 
were  employed  whenever  possible.  Duplicate  determinations  were 
made  in  all  cases  and  in  some  triplicate  or  even  quadruplicate.  Only 
the  briefest  description  of  the  official  methods  used  is  given  below. 
For  a  detailed  description  reference  may  be  made  to  Bulletin  107  of  the 
Bureau  of  Chemistry,  United  States  Department  of  Agriculture. 

Specific  gravity. — The  specific  gravity  was  determined  in  small  pyk- 
nometer  bottles  at  the  temperature  of  boiling  water. 

Melting  point. — The  melting  point  was  determined  according  to 
Wiley's  method,  by  placing  a  disk  of  the  fat  in  a  large  test  tube  con- 
taining boiled  distilled  water  and  boiled  alcohol  which  had  been 
cooled,  the  tube  being  placed  in  a  beaker  of  water  which  was  slowly 
heated,  and  noting,  by  means  of  a  thermometer  graduatetl  to  0.1° 
C,  the  temperature  at  which  the  disk  assumed  the  form  of  a  sphere. 

Bffractive  index. — The  refractive  index  was  determined  with  a 
Zeiss- Abbe  refractometer  which  had  been  standardized  with  distilled 
water.  The  fat  was  kept  at  a  constant  temperature  above  its  melt- 
ing point  during  the  determination  by  means  of  a  current  of  warm 
water  circulating  through  the  instrument.  The  reading  was  reduced 
later  by  means  of  a  factor  to  25°  C. 

Volatile  acids. — The  method  of  Reichert,  modifietl  by  Meissl.  was 
employed  in  the  estimation  of  the  volatile  acids,  and  the  results  are 
given  as  Reichert -Meissl  numbers.  In  saponifying,  use  was  made  of 
the  LefTman-Beam  methotl,  in  which  the  fat  is  saponified  in  a  flask 


METHODS    OF   ANALYSIS.  11 

over  a  naked  flame  with  a  mixture  of  a  strong  aqueous  solution  of 
caustic  soda  and  pure  glycerol.  The  soap  obtained  in  this  way  is 
decomposed  by  sulphuric  acid  and  the  liberated  volatile  acids  are 
distilled  over  with  the  steam  and  titrated  with  decinormal  barium 
hydroxid  solution. 

Saponification  value. — The  saponification  value,  or  Koettstorfer 
number,  was  determined  by  the  regular  ofll^cial  method.  The  fat  is 
saponified  in  a  flask  on  a  steam  bath  with  an  alcohohc  potash  solution, 
using  a  long  glass  tube  as  reflux  condenser. 

lodin  absorption  numher. — The  method  of  Hiibl  was  employed  in 
this  case.  The  fat  is  dissolved  in  chloroform  and  subjected  to  the 
action  of  a  mixture  of  an  alcoholic  solution  of  iodin  and  of  mercuric 
chlorid  in  a  dark  place  for  three  hours,  at  the  end  of  which  time  the 
unabsorbed  iodin  is  determined  by  titrating  with  standard  sodium 
thiosuphate  solution.  The  percentage  of  iodin  absorbed  by  the  fat  ia 
expressed  in  the  results  as  the  iodin  number. 

Insoluble  jatty  adds. — For  this  determination  the  official  method, 
or  that  of  Hehner,  as  it  is  called,  was  employed.  It  was  found,  how- 
ever, that  better  results  were  obtained  by  increasing  the  amount  of- 
fat  to  10  grams,  as  suggested  by  Brown."  The  results  are  given  as 
Hehner  numbers. 

COLOR    DETERMINATIONS. 

The  color  determinations  were  made  with  the  Lovibond  tintom- 
eter, using  the  standard  color  glasses.  Wliile  this  instrument  is 
not  admirably  adapted  to  the  work  with  milk  and  cream,  so  far 
as  the  authors'  knowledge  goes,  it  is  the  best  that  is  available. 
With  the  melted  fat  where  the  light  is  transmitted  tlirough  a  consider- 
able layer  the  determinations  can  be  made  quite  accurately,  but  with 
the  opaque  milk,  cream,  and  butter,  where  the  light  must  be  reflected 
from  the  surface,  very  slight  differences  in  color  can  not  be  deter- 
mined. The  results  are  given  in  terms  of  the  numbers  assigned  to 
the  standard  color  glasses. 

MICROSCOPICAL    WORK. 

In  the  microscopical  part  of  the  work  the  method  devised  by  Dr. 
S.  M.  Babcock''  was  used.  The  milk  is  diluted  with  distilled  water 
to  fifty  times  and  the  cream  to  one  hundred  times  its  volume. 
Fine  capillar}'  tubes  are  drawn  out  from  larger  tubes,  care  being  taken 
tiiat  the  resulting  tubes  are  round  in  cross  section.  They  are  then 
broken  into  pieces  2  to  3  centimeters  in  length,  and  by  means  of 
forceps  each  inhe  is  filled  by  immersing  one  end  in  the  diluted 
cream  or  milk.     The  tube  fills  instantly  by  capillary  attraction. 

"  Rt'port  of  Penn.s>lvania  State  CoUegp.  1899-1900,  p.  211. 

f>  Fourth  Annual  Report  of  the  New  York  Agricultural  E.xi)eriinent  Station,  1885. 


12 


LARGE  AND  SMALL   FAT   GLOBULES   IN    COWS     MILK. 


The  two  ends  are  then  sealed  with  vaseline.  Three  such  tubes  are 
used  for  each  sample,  and  these  are  laid  parallel  on  a  slide.  A  drop 
of  glycerin  is  then  placed  over  the  tubes  and  a  cover  glass  applied. 
The  slide  thus  prepared  is  allowed  to  remain  on  a  leveled  slab  for  half 
an  hour,  the  purpose  of  this  being  to  allow  the  globules  to  rise  to  the 
top  of  the  capillary  tubes.  At  the  end  of  thirty  minutes  the  slide  is 
placed  under  a  microscope  provided  with  an  ocular  micrometer  and  a 
mechanical  stage.  The  number  of  globules  in  50  divisions  of  the 
micrometer  scale  are  counted  in  three  places  in  each  tube,  the  inter- 
nal diameter  of  the  tube  at  each  place  of  counting  being  first  deter- 


Fio.  1.— Showing  method  of  counting  globules. 

mined  by  throwing  the  ocular  micrometer  scale  afcross  the  tube  at 
right  angles  and  carefully  counting  the  divisions  within  the  two 
walls.  The  total  number  of  globules  in  the  50  divisions  is  recorded, 
as  well  as  the  number  under  one  division  in  diameter ;  also  the  num- 
ber between  one  and  two  divisions  in  diameter,  and  the  number 
having  a  diameter  greater  than  two  divisions.  A  1-inch  ocular  and 
one-sixth  inch  objective  are  employed,  and  with  this  combination  the 
value  of  each  division  of  the  ocular  micrometer  with  the  instrument 
used  is  0.00258  millimeter.  In  this  way  nine  determinations  are 
made  for  each  sample. 


METHOD    OF    COUNTING   GLOBULES.  13 

In  order  to  compare  results,  the  number  of  globules  which  would  be 
found  in  a  standard  tube  100  divisions  in  diameter  and  50  divisions 
in  height  is  calculated  from  the  number  in  each  observed  tube  and  the 

average  taken.     In  making  this  calculation  the  formula       '^ —  is 

used.®  With  the  ocular  micrometer  used  in  this  work  the  standard 
tube  would  have  a  volume  of  0.006744  cubic  millimeter,  and  if  the 
milk  is  diluted  to  fifty  times  its  volume,  0.006744  divided  by  50,  or 
0.00013488  cubic  millimeter,  would  be  the  volume  of  the  original  milk 
contained  in  the  standard  tube.  In  the  case  of  cream  where  it  is  diluted 
to  one  hundred  times  its  volume  one-half  this  figure  represents  the 
volume  of  original  cream  in  the  standard  tube.  To  find  the  number 
of  gl.obules  present  in  0.0001  cubic  millimeter  it  is  only  necessary  to 


Fio.  2.— Showing  the  average  relative  size  of  large  and  small  fat  globules. 

multiply  the  number  in  the  standard  tube  by  the  factor  0.7414, 
obtained  by  dividing  0.0001  by  0.00013488. 

The  relative  size  is  found  by  dividing  the  percentage  of  fat  by  the 
number  of  globules  in  0.0001  cubic  millimeter  of  the  milk  or  cream. 
To  avoid  fractions,  the  figure  obtained  in  this  way  is  multiplied  by 
10,000. 

An  example  illustrating  the  method  of  calculating  may  be  given. 
A  sample  of  milk  containing  4  per  cent  of  fat  is  chosen.  I^t  30  repre- 
sent the  number  of  globules  counted  in  50  divisions  of  a  tube  whose 

a  The  volume  of  two  cylinders  having  the  same  height  but  different  diameteni  are 
to  each  other  as  the  squares  of  their  diameters.  If  we  let  n  equal  the  number  of  glob- 
ules in  the  observed  tube,  n''  the  number  in  the  standard  tube,  d  the  diameter  of  the 
observed  tube,  and  100  the  diameter  of  the  standard  tube,  then  n:n'':  :d  :  10,000,  or  n" 

I    10,000  n 
equals  tril^p—'. 

a 


14 


LARGE   AND   SMALL   FAT   GLOBULES    IN    COWS'  MILK 


internal  diameter  has  been  found  to  be  40  divisions.  Applying  the 
formula  1^'^-5L1\  we  have  -J-^^-  =187,  or  the  number  of  globules 

Avhich  would  be  found  in  a  standard  tube  whose  length  is  50  divisions 
and  whose  diameter  is  100  divisions.  Multiplying  187  by  the  factor 
0.7414,  we  have  138,  which  would  be  the  number  of  globules  in  0.0001 
cubic  millimeter.  Dividing  4  by  138  and  multiplying  by  10,000,  we 
have  290  as  the  relative  size  of  the  fat  globules  in  the  example  taken. 
As  Doctor  Babcock  states,  the  relative  size  is  not  strictly  accurate, 
since  j>ercentages  by  weight  have  been  confused  with  volume.  To 
be  strictly  accurate  we  must  multiply  the  relative  size  by  a  factor 
obtained  by  dividing  the  specific  gravity  of  the  milk  by  the  specific 
gravity  of  the  butterfat.  However,  since  relative  values  only  are 
required  in  our  work,  it  was  decided  that  the  relative  size  would 
answer  the  purpose,  so  our  calculations  have  stopped  at  that  point 
and  the  results  are  expressed  in  terms  of  relative  size. 

RESULTS    OF    EXPERIMENTS. 

The  following  tables  present  data  as  to  source  of  samples  and 
results  of  the  experiments: 

Table  I.— Data  concerning  source  of  samples. 


Sample 
No. 


Date. 


Breed  of  cow. 


Date  of 

last 
calving. 


Time 

in 
milk. 


Age  of 
cow. 


DaUy 
yield  of 

milk 

when 

sample 

was 

taken. 


Average 

fat 
content. 


1906 
Sept.  25 

Oct.   3 ; 

Oct.     9 
Oct.   28 


1908. 
Jan.    17 

Jan.   20 

Jan.    23 

Jan.   26 

Feb.  n 
Feb.  21 


1906. 

Ayrshire !  July   10 

do j  Aug.  13 

do. 


July   29 
Jersey I  May     8 


do Aug.  19 

....do May     8 

....do Dec,  15 

do Jan.   28 

....do Feb.    7 

— do Dec,    6 

do Jan.   25 

do Feb.    6 

Holstein Apr.   18 

do Apr.  29 

do May   13 

....do Feb.  11 


J  Shorthorn. 
1....do 

do 

do 


I  Ayrshire. 
\ do 

{Holstein.. 
....do.... 
....do.... 
(....do.... 
v. ..do.... 

py^/o-.::: 


1907, 
S^t.30 
Oct.  13 
Sept.  30 
Oct.  13 
Dec.  27 
Sept.  27 
July  17 
May  31 
July  20 
Aug.  13 
May  7 
June  16 
June  12 


Dayt. 

77 
43 
58 
151 
45 
148 
298 
2.T6 
244 
307 
257 
345 
193 
182 
168 
259 


109 

96 
112 
99 
27 
118 
192 
240 
190 
182 
279 
2.50 
254 


Yrs.Moi. 


2 

2  1 

1  I 

0 
11 

7 

3 
11 

7 

0 

4 

6 

5 

4 


Pounds. 
21.5 
28.9 
25.1 
27.9 
3a3 
22.1 
ia3 
11.1 
ia3 
7.4 
7.5 
9.1 
32.2 
20.6 
31.9 
23.1 


19.7 
18.9 
2a5 

ia2 

3L9 
22.1 
24.2 
24.9 
2a8 
3L2 
2&3 
1&5 
2&1 


Per  cent. 
4.0 

a9 

4.0 
4.5 
4.1 
5.0 
5.6 
5.2 
5.8 
6.7 
5.5 
5.0 
12 

ao 

14 

ao 


19 

4.1 

19 

4.1 

19 

175 

14 

13 

12 

11 

10 

6.48 

5.86 


RESULTS    OP    EXPERIMENTS. 


15 


Table  2.— Results  of  microscopical  work. 


Sample 
No. 


Size  of  globules. 


SmaU 
Large 
Small 
Large 
'Small 
Large 
(Small 
iLarge 
'Small 
Large 
Small 
Large 
Small 
Large 
Small 
Large 
Small 
Large 
Small 
Large 


I   Percentage 

Relative    ;    of  globules 

size  of        less  than  one 

globules.    I    division  In 

diameter. 


Percentage 
of  globules 
between  one 
and  two  di- 
visions in 
diameter 


161 
631 

91.6 
741 
112 
698 

51.7 
316 

21.7 
590 

66.8 
530 

1Z9 
703 

44.2 
417 

42.3 
299 

63.5 
691 


58.3 
15.6 
45l9 
13.8 
7a  2 
3L6 
57.8 

las 

61.2 

14.6 

67.0 

7.3 


4a4 
38.4 
49l3 
3a3 
26.6 
61.1 
4L9 
69.9 
38.5 
80.0 
32.3 
70.9 


Percentage 
of  globules 
over  two  di- 
visions in 
diameter. 


1.3 
4&0 

4.8 

46.9 

02 

7.3 

a3 
ia8 
a3 

5.4 

0.7 

21.8 


Table  3.— Results  of  chemical  work. 


Sample 
No. 


Size  of  globules. 


iSmall... 
Large.. 
SmaU.. 
Large.. 
Small.. 
Large. . 
Small.. 
Large.. 
Small  a. 
Large.. 
Small  a. 
Large. . 
SmaU.. 
Large.. 
SmaU.. 
Large.. 
SmaU.. 
Large.. 
Small.. 
I/arge.. 


Specific 
gravity. 


Melting 
pointy 


10 


a  9078 
.9044 
.9038 
.9033 
.9038 
.9035 
.9053 
.9044 
.9014 
.9014 
.9059 
.9065 


33.47 
3a  33 
3a  50 
3a  38 
3a  37 
3a  37 
32.37 
32.90 
3L85 
32.43 
32.43 
32.90 


Refrac- 

Koettstor- 

tive  in- 

fer num- 

dex. 

ber. 
23L2 

1.4609 

L4609 

23a6 

1.46C8 

229.8 

L4608 

230.6 

1. 4615 

234.0 

1. 4613 

234.6 

1. 4614 

22&3 

1.4610 

22&0 

L4565 
L  4.563 

232.4 

L4564 
L4.TC2 

23L0 
23a  3 

L  4.567 

229.6 

L4568 

22&5 

1. 4575 

226.6 

1. 4574 

226.3 

1. 4575 

227.6 

1. 4573 

227.9 

1.4.561 

23a8 

1. 4.560 

234.9 

Meissl 
nmnber. 


27.29 
27.  C6 
29.76 
29.13 
25.69 
25.49 
25.69 
2a  28 


lodin 
number. 


2a  92 


24.15 
25.01 
26.06 
24.03 
24.20 
24.98 
25.90 
26.93 
27.63 


3a91 
3a  55 
31.33 
32.29 
32.86 
32.83 
3a  37 
3a  45 


Hehncr 
number. 


29.73 
29.72 
30.20 
3a  54 
31.25 
34.56 
34.39 
a5.42 
35.  44 
2a  25 
27.62 


87.20 
!>6.83 
86.83 
8&24 
87.16 
88.53 
88.35 
85.73 
85.50 
F&13 
86.83 


<>  Sample  too  small  for  complete  analysis. 
Table  4. — Color  of  butter  and  hutterfat  frovi  large  and  small  glolntles. 


Sample 

Butter. 

ButterfiBt. 

1 

YeUow. 

Red. 

Yellow. 

Red. 

fSmall 

1 

15.5 
15.5 
15.5 
15. 5 
27.2 
27.2 
20.0 
30.0 
11.0 

ia7 

22.0 
21.0 

1.5 

[Large 

1 

1.5 

JSmaU 

1 

1.5 

Large 

1 

1.7 

ISmall 

Laras 

0.9 
.9 
.8 
.8 
.7 
.7 

LO 
.0 

0.9 
.9 
.8 
.8 
.8 
.7 
.8 
.9 

1.1 
1.6 

iSmall 

Sffl:::;;:::;;:::::;;::;::;::::;:::::;::::::::::::: 

1.5 
1.5 

.5 

Large 

.3 

iSmfll 

1.2 

10 

Lar^ 

1.0 

16  LARGE   AND  SMALL  FAT   GLOBULES   IN    COWS'  MILK. 

CONCLUSION. 

It  is  impossible  by  any  known  process  to  effect  a  complete  separa- 
tion of  the  large  and  the  small  fat  globules  in  milk.  The  best  that 
can  be  expected  is  to  secure  two  creams  from  the  milk,  one  of  which 
contains  a  large  percentage  of  large  globules  and  the  other  a  large 
percentage  of  small  globules,  or,  in  other  words,  one  in  which  the 
relative  size  of  the  fat  globules  is  comparatively  large  and  another  in 
which  it  is  comparatively  small.  That  such  has  been  obtained  in 
this  investigation  will  be  shown  by  Table  2.  A  study  of  Table  3  will 
fail  to  reveal  any  considerable  differences  between  the  fat  from  the 
large-globule  cream  and  that  from  the  small-globule  cream.  The 
differences  in  all  cases  are  small  and  in  most  cases  well  within  the 
limits  of  legitimate  experimental  error.  Table  4  also  shows  little  or 
no  variation  in  color. 

The  investigation,  of  course,  could  be  extended  by  introducing 
more  animals  and  more  samples,  but  the  range  covered  by  the 
animals  selected  and  the  number  of  determinations  made  would  seem 
to  be  sufficient  to  warrant  the  conclusion  that  in  homogeneous  milk 
the  large  and  small  fat  globules  have  identical  chemical  and  physical 
composition. 

O 


A     001  102  567     3 


